Ustilago maydis Rho1 and 14-3-3 Homologues Participate in Pathways Controlling Cell Separation and Cell Polarity

Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3ɛ and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.Morphological switching is a unique attribute of all dimorphic fungi, which alternate between budding and filamentous growth. In some cases, as with mating, this is a prerequisite for genetic diversity for this subfamily of fungi. In addition, many dimorphic fungal pathogens rely on this ability in order to effectively invade their host. In general, the transition between these alternate life forms means a complete turnover of cellular and proteomic components, which often involves cell cycle arrest and/or cytoskeletal rearrangement. Although the cellular proteomes associated with these two processes share many components, there are both temporal and spatial regulations that are manifested during the transitional phase (4).Temporal-spatial regulation of the proteome during the dimorphic transition requires cooperation and synchronized communication among different regulatory pathways. Two highly intricate, yet well-established, signaling cascades that regulate fungal morphogenesis are the mitogen-activated protein kinase (MAPK) (34, 46) and protein kinase A pathways (11). These signaling cascades detect and perpetuate extracellular stimuli, e.g., pheromones and nutrients, which lead to phase transitions in dimorphic fungi. Although the mechanisms are not as fully understood, members of two highly conserved families of proteins, Rho/Rac GTPases and 14-3-3 proteins, have also been shown to control filamentation. Constituents of the Rho/Rac protein family have been shown to regulate actin organization (26, 35, 36), cytokinesis (3, 49, 52), cell integrity (42, 56), pathogenicity (29), signal transduction (22, 44, 56), and cell migration (8). Their activity is dependent upon the reversible binding of guanine nucleotides catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (15, 22, 23, 25, 44). Upon activation, Rho-GTPases stimulate downstream effector proteins such as p21-activated kinases (PAKs) (29, 51) or Rho kinase (ROCK) (36). Based on in silico analysis of the genome sequence, the fungal pathogen of maize, Ustilago maydis, contains six different Rho/Rac encoding genes: cdc42, rac1, and four additional genes predicted to encode Rho-like proteins (20). Of these, only the roles of Cdc42 and Rac1 have been examined in depth. Cdc42 was shown to regulate cytokinesis, while Rac1 regulates hyphal development in U. maydis (26). We examine here the place of a Rho1 homologue, Rho1, in U. maydis cell morphology, polarity, and development.Similarly, the highly conserved, ubiquitously expressed 14-3-3 proteins that are found in most eukaryotes have also been shown to contribute to cellular differentiation and cytoskeletal organization. Like Rho-GTPases, 14-3-3 proteins play multiple roles, in cytoskeletal function, cell cycle regulation, apoptosis, and the regulation of a variety of signaling pathways (17, 19, 33, 50). These acidic proteins have been found in each cellular compartment and most organisms examined possess multiple isoforms: seven isoforms are found in mammals, and as many as fifteen isoforms have been identified in plants (31). Interestingly, the yeast Saccharomyces cerevisiae, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans, each possess only two 14-3-3 isoforms (50), while Candida albicans contains a single isoform (37). They function typically by binding their particular ligands at phosphoserine or phosphothreonine residues (50). It is not clear what 14-3-3 proteins do in the processes mentioned above, whether they act as scaffolds or effectors. Inspection of the U. maydis genome sequence revealed that this organism could be ideal for the study of 14-3-3 proteins because, unlike most other organisms, the U. maydis genome contains only a single 14-3-3 homologue. Due to its predicted binding of phosphorylated proteins, we named this homologue Pdc1 (for phosphorylation domain coupling protein [10]). Recently, the protein (also designated Bmh1 [32]) was also shown to be involved in cell cycle regulation.Despite the functional differences between Rho-GTPases and 14-3-3 proteins, we provide evidence that members of these two families participate in the same regulatory cascade(s) that control morphogenesis in the dimorphic fungus U. maydis. We are able to demonstrate that both Pdc1 and Rho1 are essential for cell viability. In addition, overexpression of Rho1 led to the reduction of filamentation. Overexpression of Rac1 triggers filamentation in U. maydis (13, 29). We show here that deleting Rac1 eliminates the lethal effect imposed by either Rho1 or Pdc1 depletion. Our results have led us to predict that both Rho1 and Pdc1 are negative regulators of Rac1 in U. maydis and that they play important roles in polarized growth and cytokinesis.